The use of recombinant host cells in the expression of heterologous proteins has in recent years greatly simplified the production of large quantities of commercially valuable proteins which otherwise are obtainable only by purification from their native sources. Currently, there is a varied selection of expression systems from which to choose for the production of any given protein, including bacterial and eukaryotic hosts. The selection of an appropriate expression system often depends not only on the ability of the host cell to produce adequate yields of the protein in an active state, but, to a large extent, may also be governed by the intended end use of the protein.
One problem frequently encountered is the high level of proteolytic enzymes produced by a given host cell or present in the culture medium. Proteases are a major problem for the production of heterologous proteins in filamentous fungi, since they are present at every stage of the secretion process as well as several extracellular proteases.
It has been suggested that one could provide host organisms deprived of the ability to produce specific proteolytic compounds. For example, International Patent Application WO 90/00192 (Genencor) describes filamentous fungal hosts incapable of secreting enzymatically active aspartic proteinase, and EP 574 347 (Ciba Geigy AG) describes Aspergillus hosts defective in a serine protease of the subtilisin-type.
Other examples of proteases which have been reported to reduce the stability of protein products include metalloproteases and alkaline proteases.
WO 1998/012300 describes fungal hosts having improved stability of heterologous protein products in which the host cell have been genetically modified to express significantly reduced levels of both a metalloprotease and an alkaline protease.
Another intracellular protease, a serine protease of the subtilisin type produced e.g. by A. niger and designated PepC has been described, the gene expressing it cloned, and a deletion mutant described in EP 574 347 and in Frederick et al., Gene, 125 57-64 (1993).
Another group of processing proteases is the kexin family of proteases, which recognizes dibasic amino acid motifs and are involved in removing N-terminal propeptides. Kexin proteases have a narrow substrate specificity. Fungal kexin like maturases have been cloned and characterized in Aspergillus niger (Jalving et al., 2000, Applied and Environmental Microbiology 66:363-368), in Aspergillus nidulans, designated KpcA (Kwon et al., 2001, Molecular Cell 12:142-147), and in Aspergillus oryzae (Mizutani et al., 2004, Eukaryotic Cell 3:1036-1048).
For heterologous production of recombinant proteins in a fungal host the choice of a suitable host resulting in the optimal expression and stability of the recombinant protein is to a large extent dependent on the protein and thus the presence of specific protease recognition sites in the protein. The presence of such recognition sites can be very difficult to predict. It is therefore desirable to identify new mutant fungal strains specifically tailored to the production of a desired protein. The present invention relates to the identification of desired protease mutations in fungal host strains useful for the production of heterologous proteins and particularly to the combination of such mutations.